Ewing's sarcoma is a very painful cancer of the bone (and, more rarely, soft tissue) that strikes children and adolescents. Despite treatment with surgery, chemotherapy, and radiation, many patients with Ewing's sarcoma do not survive, particularly if the cancer has metastasized. Recurrent Ewing's sarcoma tends to exhibit resistance to chemo- and radiotherapies, motivating the development of novel therapies, including immunotherapies. However, some Ewing's sarcoma cells can escape elimination by the immune system and the mechanisms underlying evasion by this cancer are poorly understood, although there have been reports of down-regulated expression of major histocompatibility complex (MHC) class I molecules, and correlation of decreased MHC class I expression and poor survival. Since the function of MHC class I molecules is to present antigens (including cancer-associated antigens) to cytotoxic T lymphocytes, reduction of their surface expression could cause immune evasion. We have sought to characterize cellular changes associated with Ewing's sarcoma cells that avoid immune recognition, investigating the roles of MHC molecules and amyloid precursor-like protein 2 (APLP2) in this process. APLP2 is a ubiquitously expressed member of a protein family that also includes amyloid precursor protein (APP) and the nervous system-restricted amyloid precursor-like protein 1 (APLP1). APLP2 is found in transmembrane form at the cell surface, and its cleavage by beta-secretases leads to the production of ~12 kDa C-terminal fragments. Our new data suggests that elevated expression of APLP2 in Ewing's sarcoma cell lines reduces surface expression of MHC class I molecules. Following treatment with radiation, the Ewing's sarcoma cells that had relatively low APLP2 expression had increased MHC class I expression at the cell surface, but APLP2- high Ewing's sarcoma sub-populations did not. In our preliminary experiments, we have also demonstrated that following irradiation of Ewing's sarcoma cells APLP2 reduces cell cycle arrest at G2/M and lowers the proportion of cells with sub-G1 DNA content (indicating apoptosis). On the basis of our preliminary data, our central hypothesis is that APLP2 facilitates the survival of Ewing's sarcoma cells. To test our central hypothesis, the Specific Aims of this proposed project are (1) to determine the role of APLP2 in resistance of Ewing's sarcoma cells, pre- and post-irradiation, to cytotoxicity, and (2) to determine the contribution of APLP2 to radiation resistanc by Ewing's sarcoma cells. Overall, the studies proposed in this application are expected to provide a new perspective on molecular pathways regulating Ewing's sarcoma cell survival, involving evasion of both immunity and radiotherapy.